Sleep Flashcards
What switch between sleep and wake stable states?
Due to the build-up of sleep pressure during wakefullness and the dissipation of sleep pressure during sleep
What is the sleep-wake cycle entrained to?
To the circadian cycle
When do you fall asleep?
When the sleep pressure is great
When do you wake up?
When the dissipation is sufficient
What does the switch in sleep-wake state occurs from due to?
Due to neurons that have antagonistic functions:
1) Wake promoting neurons
2) Sleep promoting neurons
What are wake promoting neurons activated by?
What do they promote?
By dissipating sleep pressure
Promote transition into wakefullness when a threshold of activation is reached
What are sleep promoting neurons activated by?
What do they promote?
By increasing sleep pressure
Promote transition into sleep when reach a certain threshold
Describe the sleep-wake transition
Rapid
Bistable
What controls the sleep-wake transition
Controlled by a network of sleep and wake neurons
Where are wake promoting neurons found in the hypothalamus?
Lateral hypothalamus
Tubomammillary nucleus
What neurons are in the lateral hypothalamus?
Neurons that synthesise and secrete OREXIN (HYPOCRETIN)
What neurons are in the tubomammillary nucleus?
Neurons that synthesise and secrete HISTIDINE
What is narcolepsy?
A chronic SLEEP DISORDER that causes:
- Drowsiness
- Excessive day-time sleepiness
- Frequent day time napping
What can narcolepsy occur with?
- Cataplexy
- Sleep paralysis
Who does narcolepsy occur in?
Young adults/adolescents
What happens to neurons in narcolepsy?
85-90% reduction in Hypocretin neurons in the lateral hypothalamus (LH)
What leads to narcolepsy?
1) Mutations in HYPOCRETIN
2) LESION or ATTACK to the TMN/lack of HISTIDINE
What does ISH of a hypocretin brain show in comparison to a normal brain?
No hypocretin in a narcoleptic brain
What is important to know about hypocretin?
The genetic programme that establishes the neuronal hypocretin+ lineage and maintains fully functional hypocretin expression
What transcription factor specifies hypocretin+ neurons?
How was this discovered?
Lhx9
Discovered by screens of zebrafish looking for factors that can specify Hct neurons:
1) Firstly seeing if could see normal Hct+ neurons - using ISH with antisense probe - saw at 24hpf and 120hpf
2) Then, made a transgenic reporter line using: RPF under the hct promoter
3) Sort RFP+ cells from dissociated cells from isolated brain using FACS
4) Microarray to identify the transcripts enriched in the Hcrt neurons
What type of TF is Lhx9?
LIM homeobox TTF
How can conclude that fluorescent cells have same expression pattern as NORMAL Hcrt expression?
Because RFP downstream of Hrct promoter - promoter on where gene is expressed
What is FACS?
Fluorescence-activated cell sorting - sorts cells if they express RFP –> RFP cells express Hcrt under the control of the endogenous promoter
How does a microarray help to detect the TF responsible for the specification of Hcrt neuorns?
Identifies genes that are up regulated in these cells compared to all other cells –> look for genes that are TF
Focus on TF that have no KNOWN function
How was it validated that the identified Lhx9 TF was expressed in the right place at the right time?
ISH of hcrt and lhx9 on either:
1) WT embryos
OR
2) tg(hcrt:EGFP)
What did the ISH on WT embryos show?
- Hcrt mRNA is UPREGULATED in the LATERAL HYPOTHALAMUS
- All hct+ neurons express lxh9
What did tg(hcrt:EGFP) show?
- OVERLAP between hcrt and lhx9 expression - ALL hcrt+ cells express lhx9
- But there some lxh9 cells that DON’T express hcrt
What shows that lhx9 is SUFFICIENT to specify Hcrt neurons?
How was over expression achieved?
What was the control?
Transient ECTOPIC overexpression of lhx9 –> enough to specify hcrt neurons
Over expression:
- Clone Lhx9 downstream of heat shock promoter and increase the temperature
Control:
- Injection of GFP and NO Lhx9
How does overexpression of lhx9 induce hct neuronal fate?
Alters the fate of the progenitor that doesn’t normally express hct to express hct and direct them down an alternative programme, where they will commit to a hct neuronal identity
What organisms has it been shown that lhx9 is sufficient to induce hct neurons?
Mouse and zebrafish
What are 2 control ‘icing on the cake’ experiments?
Show the induced ectopic neuronal cells are TRUE Hcrt+ cells:
1) Express OTHER MARKERS that are detected in endogenous hcrt+ neurons
2) Show the axons of the ectopic Hcrt+ cells project to the SAME targets as endogenous Hcrt+ neuronal axons
How test if lhx9 is necessary to induce Hcrt+ neurons?
How?
What are the results?
KNOCK DOWN Hcrt using:
1) Splice-blocking morpholoino
- Control morpholino - patch of Lhx9 expressing cells
- Lhx9 morpholino - patch of cells is gone
- -> Reduced Hct neurons
2) CRISPR/Cas9
- Cas9 alone –> Hct neurons
- Cas9 with Lhx9 guide RNA –> Knockdown
- -> Loss of Lhx9 and Hcrt neurons
How do TF act?
In the NUCLEUS in a CELL AUTONOMOUS way:
- Upregulate TF at mRNA lels
- mRNA translated in the cytoplasm
- Protein –> back into nucleus and acts on DNA - bind to enhancer of the gene and allow RNA pol to come in an upregulate transcription
What is hypothesised about Lhx9 interaction with Hct?
What is the evidence for this?
Hypothesis it DIRECTLY promotes Hcrt expression as Lhx9 is a TF
Evidence:
1) Double-flourescent ISH of hct and lhx9 on zebrafish embryos shows hcrt expression in almost ALL lhx9 over expressing cells following heat shock
2) From another study - identification of Lhx9 binding site upstream of gene
- Same sequence upstream of hcrt neurons
- Mutate binding site (lhx9 cant bind) –> failure to get hcrt transcribed
–> Directly regulate
What type of neurons are the Hcrt+ neurons?
WAKE-PROMOTING neurons
In the LATERAL HYPOTHALMUS
What neurons do the wake-promoting Hcrt+ neurons interact with?
In ANTAGONISTIC NETWORK with SLEEP-PROMOTING neurons
According to studies, what are the sleep-promoting neurons?
GABA-ergic neurons under the control of the ALTERNATIVE Lhx6 gene
- Project to wake-promoting neurons (Hct+) and GABA-ergic neurons of the LH to SUPPRESS their activity to PROMOTE SLEEP
Where are the Lhx6 GABA-ergic neurons present?
In the ZONA INCERTA
What activates the Lhx6 GABA-ergic neurons?
Sleep pressure
What does conditional KO of the Lhx6 in the developing diencephalon cause?
DECREASES in both REM and non REM sleep –> WAKEFULLNESS
How were the GABA-ergic Lhx6 cells discovered?
By tracing and studying Lhx6+ neurons using a transgenic reporter line (Lhx6-Cre:ChR2-eYFP) and optogenetics
Describe the transgenic reporter line (Lhx6-Cre:ChR2-eYFP)
Express eYFP under the control of Lhx6 promoter
eYFP conditionally activated by optogenetics
How can it be shown if a particular neuorn/circuit governs a particular behaviour?
Activate individual neurons/circuits through OPTOGENETICS
What is optogenetics?
Using light to activate a neuron expressing a particular gene
What is the discovered programme of development of the hcrt neuron so far?
Why do we want to know the full programme?
1) Shh (deriving from the hypothalamic floor-plate like cells) is secreted –> induces Lhx9+ in non-autonomous way
2) Lhx9 –> induces hcrt+ neurons in an autonomous way
Want to know full programme as there is currently NO CURE for narcolepsy and the current drugs have SEVERE side effects
What are the drugs currently used to treat narcolepsy?
What do they help with?
1) Amphetamines - help with day-time sleepiness
2) Antidepressants - supress REM, cataplexy, paralysis and hallucination
3) Hypocretin - CURED dogs of cataplexy
What is a potentially useful approach to treating narcolepsy?
CELL REPLACEMENT THERAPY to replace missing cells using Hcrt-expressing cells from own body to prevent rejection
How create iPS?
2-step process:
1) Take cells from the body and reprogram by adding TF that are CHARACTERSTIC of pluripotent cells
2) Apply ex vivo factors to the iPS that NORMALLY direct th cells in vivo –> get specialised cells
What factors are required for Hct fate?
How was this discovered?
Peg3 Ahr1 Six6 Nr2f2 Prrx1
Discovered by RNA sequencing
What are the advantages of iPS?
1) No need for embryo
2) Can get INDIVIDUAL-SPECIFIC pluripotent cells
What are the clinical applications of iPS?
- Ability to differentiate into MANY cell types
- Easy accessible
- Vastly renewable
- Individual-specific (personalised)
What is the iPS approach being used for?
To direct the differentiation of MANY types of hypothalamic neuron
